This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The t(9;22)(q34;q11) generating the BCR/ABL1 fusion gene represents the cytogenetic hallmark of chronic myeloid leukemia (CML).
About 5–10% of CML cases show variant translocations with the involvement of other
chromosomes in addition to chromosomes 9 and 22. The molecular bases of biological
differences between CML patients with classic and variant t(9;22) have never been
clarified.

Findings

In this study, we performed gene expression microarray analysis to compare CML patients
bearing variant rearrangements and those with classic t(9;22)(q34;q11). We identified
59 differentially expressed genes significantly associated with the two analyzed groups.
The role of specific candidate genes such as TRIB1 (tribbles homolog 1), PTK2B (protein tyrosine kinase 2 beta), and C5AR1 (complement component 5a receptor 1) is discussed.

Conclusions

Our results reveal that in CML cases with variant t(9;22) there is an enhancement
of the MAPK pathway deregulation and show that kinases are a common target of molecular
alterations in hematological disorders.

Keywords:

Background

Chronic myeloid leukemia (CML) is a myeloproliferative disorder derived from hematopoietic
stem cell transformation and characterized by heterogeneous biological and clinical
features. The CML molecular marker is BCR/ABL1 fusion gene generation as a consequence of a reciprocal t(9;22)(q34;q11)
[1,2]. In most cases, the Philadelphia (Ph) chromosome is cytogenetically detectable but
about 5–10% of CML patients show variant t(9;22)(q34;q11) rearrangements with the
involvement of additional chromosomes
[3,4]. In these cases the BCR/ABL1 fusion gene can be revealed by Fluorescence in situ hybridization (FISH) or reverse
transcriptase-polymerase chain reaction
[5]. The occurrence of genomic microdeletions proximally to ABL1 or distally to BCR has been reported in CML cases with variant translocations with a greater frequency
(30-40%) than in cases with classic t(9;22) (10-18%)
[6,7]. The prognostic significance of variant t(9;22) was unclear and debated in the pre-imatinib
era, whereas recent studies of large CML series have reported that the presence of
variant translocations has no impact on the cytogenetic and molecular response or
on prognosis
[6,8]. However, the molecular bases of biological differences between CML patients with
classic and variant t(9;22) have never been elucidated.

In this study, we performed gene expression profiling (GEP) by microarrays to identify
a signature discriminating CML patients bearing variant rearrangements from those
with classic t(9;22)(q34;q11). A list of 59 genes was found to be significantly associated
with the two analyzed groups showing a differential expression. We applied network
analysis to evaluate potential pathways involved in CML heterogeneity. An overall
deregulation of genes encoding for protein kinases and involved in crucial cellular
pathways such as MAPK (mitogen-activated protein kinase) signaling was found, unveiling
the biological basis of differences in the CML patients subgroup with variant rearrangements.

Findings

Banding and molecular cytogenetic analyses allowed the identifications of 12 CML cases
with classic t(9;22) and 8 cases with variant translocations (Additional file
1, Additional file
2). The BCR/ABL1 fusion analysis revealed the occurrence of b2a2 or b3a2 junctions in 10 (7 with
classic and 3 with variant rearrangement) and in 10 (5 with classic and 5 with variant
rearrangement) cases, respectively. All these patients were selected for further GEP
analysis by oligonucleotide microarrays (Additional file
2). A set of 59 genes was identified as differently expressed in CML cases with variant
t(9;22) rearrangements. All deregulated genes showed a more than 2 fold expression
change; results of gene expression analysis indicated that 45 out of 59 differentially
expressed genes were up-regulated whereas 14 were down-regulated (Figure
1; Additional file
3).

Involvement of kinases in the RAS/MAPK pathway

Further Ingenuity Pathways Analysis (IPA) analysis yielded strong indications that
19 out of 59 dysregulated genes from our dataset are involved in the “Haematological
System Development and Function, Tissue Morphology, Cellular Development” network
(Figure
2A). A central role in this network is played by several proteins that are known to
be activated in BCR/ABL1 cells, namely ERK1/2 (extracellular signal-regulated kinases), p38MAPK (p38 mitogen-activated
protein kinase), JNK (c-Jun N-terminal kinase), and cell cycle regulator AKT (RAC-alpha serine/threonine-protein kinase) that have a key function in multiple cellular
processes such as apoptosis and cell proliferation (Figure
2A). In this respect, three main cellular processes are dysregulated by the BCR/ABL
oncoprotein: RAS/MAPK which induces activation of proliferation, the PI3K (phosphatidylinositol-3
kinase)/AKT that activates apoptosis, and JAK/STAT which leads to an increased transcriptional
activity
[9]. Noteworthy, the upregulated kinase genes, previously revealed by DAVID analysis,
are also enclosed in the network identified by IPA and establish direct or indirect
interactions with other network components (Figure
2A). Moreover, TRIB1, PTK2B and C5AR1 kinases are involved in the regulation of the
RAS/MAPK pathway (Figure
2B)
[10-12]. TRIB1 is a signaling regulatory protein involved in leukemogenesis by regulating
cellular proliferation and myeloid differentiation
[10]. This protein binds to the ‘middle layer’ of kinases in the MAPK network, MAPKK (mitogen
activated protein kinase kinase), and acts as an adaptor between the MAPKK pathway
and C⁄EBPα (CCAAT⁄enhancer binding protein alpha). In fact, TRIB1 interacts with MEK1
(mitogen-activated ERK kinase 1) and MKK4 (MAP kinase kinase 4) and enhances phosphorylation
of ERK1⁄2, promoting cell proliferation and suppressing apoptosis. ERK1/2 phosphorylation
is required for C⁄EBPα degradation and the activation of hnRNP-E2 (poly(rC) binding
protein 2), a C⁄EBPα repressor (Figure
2B)
[10]. Moreover, PTK2B is a proline–rich kinase involved in calcium induced regulation
of ion channel and activation of the MAPK signaling pathway by stimulating JNK and
ERK1/2 activity (Figure
2B)
[11]. PTK2B is a member of the FAK tyrosine kinases family that could be activated by
BCR-ABL causing an aberrant cell adhesion. C5AR1 is a member of the rhodopsin family
of G protein-coupled receptors and is also the receptor of C5a anaphylatoxin, a strong
proinflammatory mediator. It has been recently demonstrated that C5a binding to C5aR
on human intestinal epithelial cells activates ERK1/2 and AKT phosphorylation
[12].

Figure 2.Deregulated genes in CML cases with variant t(9;22). (A) “Hematological System Development and Function, Tissue Morphology, Cellular Development”
network deriving from GEP in CML cases with variant t(9;22). Both direct (solid lines)
and indirect (dashed lines) interactions among genes are shown. Colored symbols correspond
to genes included in our set of differentially expressed genes (red = upregulated
and green = downregulated). (B) The involvement of TRIB1, PTK2B and C5AR1 kinases in the RAS/MAPK pathway downstream
to the BCR/ABL oncoprotein.

To date several profiling studies in CML have been reported focusing mainly on predicting
response to imatinib therapy and identifying a gene-specific signature for different
stages of the disease
[13,14]. Our GEP analysis performed on CML cases with variant t(9;22) may improve the understanding
of the biological mechanisms at the basis of the CML heterogeneity. Overall, our results
reveal that in CML cases with variant t(9;22) there is an enhancement of the MAPK
pathway deregulation already known to underlie the CML pathogenesis and point out
the role of interesting candidate genes, such as TRIB1, PTK2B, and C5AR1. These findings show that kinases are a common target of molecular alterations in
hematological disorders and reinforce the idea that a perturbed action of signal transduction
pathways is one of the hallmarks of cancer.

Competing interest

The authors declare that they have no competing interests.

Authors’ contributions

FA, AZ, and LA contributed to the overall experimental design and wrote the manuscript.
NC and GT conducted FISH experiments. PC and LI performed banding cytogenetics. ARR
contributed to clinical data collection. AM and FCM contributed to molecular analysis
experiments. FA and GS supervised the manuscript preparation and gave the final approval.

Acknowledgments

The authors would like to thank Ms. MVC Pragnell, B.A. for language revision of the
manuscript.